WO2023085342A1 - ＳａｐＢタンパク質の修飾体、ＳａｐＢタンパク質およびその修飾体の調製方法、並びにＳａｐＢタンパク質またはその修飾体を溶解した水溶液 - Google Patents

ＳａｐＢタンパク質の修飾体、ＳａｐＢタンパク質およびその修飾体の調製方法、並びにＳａｐＢタンパク質またはその修飾体を溶解した水溶液 Download PDF

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WO2023085342A1
WO2023085342A1 PCT/JP2022/041835 JP2022041835W WO2023085342A1 WO 2023085342 A1 WO2023085342 A1 WO 2023085342A1 JP 2022041835 W JP2022041835 W JP 2022041835W WO 2023085342 A1 WO2023085342 A1 WO 2023085342A1
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sapb
protein
modified
composition
urea
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French (fr)
Japanese (ja)
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修一 廣瀬
政敬 光本
恵介 松山
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Nagase and Co Ltd
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Nagase and Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

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  • the present invention relates to a modified form of SapB protein, a method for preparing SapB protein and modified form thereof, and an aqueous solution in which SapB protein or modified form thereof is dissolved.
  • the SapB protein was discovered as a protein having surface activity (Non-Patent Document 1).
  • the SapB protein is generated by cleavage from its precursor, RamS.
  • SapB is part of RamS (SapB precursor) and is encoded by the ramS gene.
  • the ramS gene resides within an operon that includes ramC, ramS, ramA, ramB, and ramR.
  • Non-Patent Document 2 contains dehydroalanine and lanthionine bonds. This modified peptide can be totally synthesized (Non-Patent Document 3).
  • the present invention provides a modified SapB protein, a method for preparing the SapB protein and its modified body, and an aqueous solution in which the SapB protein or its modified body is dissolved.
  • the present inventor found that extraction of SapB protein from SapB protein-expressing cells at room temperature in the presence of urea or under heating conditions improved the recovery rate of native SapB protein.
  • the present inventors also discovered a new modified SapB chemically modified at the N-terminus during the purification of the SapB protein. Specifically, when the present inventors extracted SapB protein from SapB protein-expressing cells under heating conditions and in the presence of urea, they were able to obtain a new modified SapB chemically modified at the N-terminus in high yield. and that the modified SapB obtained has good surfactant and emulsifying properties.
  • the new modified SapB chemically modified at the N-terminus was stable against some degradation treatments.
  • the present inventors have further found that the poorly water-soluble SapB protein and its modifications exhibit high solubility in alkaline buffers.
  • the present inventors have further found that the modified SapB protein exhibits high solubility in pH 6 or higher buffers.
  • a method for preparing a SapB protein comprising: providing a cell that produced a SapB protein; Cells (a) under a temperature condition equal to or higher than a first predetermined temperature and in the absence of urea; (b) at a temperature below a second predetermined temperature and in the presence of urea; or (c) at a temperature at or above a third predetermined temperature and in the presence of urea ⁇ wherein the first The predetermined temperature is 40° C.
  • a method including (2) The method according to (1) above, wherein the incubation is performed under (a) a temperature condition equal to or higher than a first predetermined temperature and in the absence of urea. (3) The method according to (1) above, wherein the incubation is performed under (b) a temperature condition below a second predetermined temperature and in the presence of urea. (4) The method according to (1) above, wherein the incubation is performed under (c) a temperature condition equal to or higher than the third predetermined temperature and in the presence of urea.
  • a surfactant comprising the modified SapB protein according to any one of (5) to (9) above or the composition according to (10) above.
  • An emulsifier comprising the modified SapB protein according to any one of (5) to (9) above or the composition according to (10) above.
  • a composition comprising an emulsion containing a fat-soluble solute molecule and the modified SapB protein according to any one of (5) to (9) above.
  • composition according to (14) above which is a cosmetic, a household detergent, or a food or drink.
  • a composition comprising an isolated SapB protein or modification thereof and an aqueous solution, A composition, wherein an isolated SapB protein or modification thereof is dissolved in an aqueous solution, the aqueous solution being alkaline.
  • the composition according to item 14, wherein the modified form of isolated SapB is the modified form according to any one of (5) to (9) above.
  • the composition according to (16) or (17) above, wherein the aqueous solution has a pH of 8 or higher.
  • the composition according to any one of (16) to (18) above, wherein the aqueous solution has a pH in the range of 8-11.
  • An aqueous composition comprising a modified isolated SapB protein and having a pH of 6 or greater.
  • a SapB expression vector comprising a gene expression cassette having part or all of the SapB operon region.
  • SapB expression vector 3.
  • a composition comprising the SapB expression vector of (31) or (32) above.
  • (38) A method for preparing a SapB protein comprising: introducing the SapB expression vector according to (31) or (32) above into a cell; culturing the resulting cells to produce SapB protein; method including.
  • (39) A method for preparing a SapB protein comprising culturing the cell according to (33) or (34) above to produce the SapB protein.
  • a method for preparing a modified SapB protein comprising: introducing the SapB expression vector according to (31) or (32) above into a cell; culturing the resulting cells to produce SapB protein; heating the resulting SapB protein in the presence of urea to obtain a modified SapB protein; method including.
  • (41) A method for preparing a modified SapB protein comprising: culturing the cells according to (33) or (34) above to produce the SapB protein; heating the resulting SapB protein in the presence of urea to obtain a modified form of SapB; method including.
  • the method for preparing SapB protein of the present invention is suitable for recovering poorly water-soluble SapB protein expressed in cells.
  • Modified forms of the SapB protein of the present invention are stabilized by modification and can be resistant to amino acid degradation from the N-terminus.
  • FIG. 1 shows the emulsifying action of SapB with new chemical modifications obtained in this example.
  • FIG. 2A shows high-performance liquid chromatographs of native SapB protein and SapB with new chemical modifications obtained in this example.
  • FIG. 2B shows high-performance liquid chromatographs of native SapB protein and SapB with the new chemical modification obtained in this example (modified SapB protein) in solutions incubated for different times in the presence of different concentrations of urea. show.
  • FIG. 3A shows the separation of SapB protein by liquid chromatography and the respective MS extracted chromatographs.
  • FIG. 3B shows the mass spectrometry spectrum of native SapB and the mass spectrometry spectrum of SapB with new chemical modifications obtained in this example.
  • FIG. 3C shows enlarged views near m/z 130 to 160 of the peaks of natural SapB (upper row) and NH 2 -C(O)-modified SapB (lower row), respectively.
  • FIG. 3D shows enlarged views near m/z 960-1190 of the cleaved spectra of natural SapB (upper row) and SapB modified with NH 2 —C(O)— (lower row).
  • FIG. 4 shows the results of SDS-PAGE of suspensions (left panel) and centrifugation supernatants (right panel) in buffers with different pHs containing modifications of the SapB protein. Arrowheads in the figure indicate the band of the modified SapB protein.
  • FIG. 5 shows high performance liquid chromatographs of centrifugation supernatants of buffers with different pH containing native SapB protein. Arrowheads in the figure indicate peaks of modified SapB proteins.
  • FIG. 6 shows high performance liquid chromatographs of centrifugation supernatants of buffers with different pHs containing modifications of the SapB protein. The arrow in the figure indicates the peak of the modified SapB protein.
  • FIG. 7 shows the expression of various SapB proteins. Lane 1 is SEQ ID NO: 9, Lane 2 is SEQ ID NO: 7, Lane 3 is SEQ ID NO: 5, Lane 4 is SEQ ID NO: 6, Lane 5 is SEQ ID NO: 8, Lane 6 is 0.1% SapB protein (lyophilized). handle. The arrow in the figure indicates the position of the SapB protein band.
  • FIG. 8 shows the relationship between the SapB concentration and the surface tension (mN/m) according to the Wilhelmy method.
  • FIG. 9 shows the effect of surface processing by SapB application processing.
  • Isolate or “isolate” means to separate a particular substance or molecule from at least one or more other substances or molecules. Isolating may include separating a particular substance from a cell, for example, when the cell is induced to produce the substance. Isolation is also, after isolation, separation of liquid phase and solid phase by filtration, solution exchange by dialysis, separation by molecular size such as gel filtration, separation by hydrophobic column, separation of specific molecules based on affinity, etc. may be purified.
  • Isolation is a different concept from purity, but the isolated specific substance is, for example, 50 wt/wt% or more, 60 wt/wt% or more, 70 wt/wt% or more, 80 wt/wt% or more , 90% w/w or higher, or 95% w/w or higher. Purity can be determined as weight/weight percent of the specified substance relative to the total solutes if the specified substance is present in the solvent.
  • N-terminus is the amino terminus of polypeptides and proteins.
  • Polypeptides and proteins are macromolecules in which amino acids having an amino group and a carboxyl group are linked by dehydration condensation, and the polypeptides and proteins have a carboxyl group and an amino group at both ends.
  • the terminal on the carboxyl group side is called the C-terminal
  • the terminal on the amino group side is called the N-terminal.
  • the N-terminal amino group is different from the side chain amino group of lysine (or ornithine) and the side chain guanidino group of arginine in that it is an amino group capable of forming a peptide skeleton.
  • an N-terminal amino acid refers to the amino acid at the tip of the N-terminus (the first amino acid).
  • a terminal amino group has an amino group attached to the alpha carbon of the terminal amino acid.
  • amino acid means an ⁇ -amino acid that is a building block of proteins in living organisms.
  • An ⁇ -amino acid has at least an amino group and a carboxyl group on the ⁇ carbon, and the amino group and the carboxyl group undergo dehydration condensation to form a peptide bond to form a polypeptide or protein.
  • Amino acids include alanine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, tyrosine, aspartic acid, asparagine, glutamic acid, and serine.
  • amino acids exist in D-type and L-type (exceptionally, glycine does not have an asymmetric carbon and thus does not have an optical isomer), naturally occurring amino acids are basically L-type.
  • a side chain in an amino acid means a moiety other than the above amino group and carboxyl group attached to the ⁇ -carbon and hydrogen.
  • proline is originally a molecule classified as an imino acid, it is treated as an amino acid in biochemistry and is also treated as an amino acid in this specification.
  • dehydroalanine a dehydroamino acid with a non-natural structure, is found, and dehydroalanine is also classified as an amino acid.
  • Dehydroalanine has the following structure: have
  • a "lanthionine bridge” or “lanthionine bond” is a bridge or bond by two alanine residues forming a thioether bond between the ⁇ -carbons.
  • Two alanine residues forming a thioether bond at the ⁇ -carbon have the formula HOOC--CH( NH.sub.2 )-- CH.sub.2 --S-- CH.sub.2 --CH( NH.sub.2 )--COOH.
  • the carbons on both sides of S are ⁇ carbons.
  • sapB protein what was a serine and cysteine residue before translational modification undergoes dehydration condensation between the side chains to form a structure of two alanine residues forming a thioether bond at the ⁇ carbon.
  • protecting refers to chemical modification (also simply referred to as “modification”) in which a reactive group is modified with a protective group to convert it into an inactive functional group. . Removing a protecting group from a protected functional group is called deprotection. When it is not necessary to remove the protecting group, it can be used with the protecting group still present.
  • emulsion refers to a dispersion solution containing a dispersoid and a dispersion medium, and in which both the dispersion medium and the dispersoid are liquid. Emulsions are also called emulsions or emulsions. As used herein, emulsification refers to bringing two separate liquids into an emulsion. In this specification, a substance having an emulsifying action is referred to as an emulsifier. Immiscible liquids, such as water and oil, can form micelles when mixed with the addition of an amphiphilic substance (also called a surfactant). It is known that amphiphilic substances form micelles to stabilize droplet dispersions.
  • an amphiphilic substance also called a surfactant
  • Emulsions include, for example, oil-in-water (O/W) emulsions and water-in-oil (W/O) emulsions.
  • Dispersion media include, for example, aqueous solvents such as water, such as distilled water.
  • the term "SapB protein” refers to a protein (RamS) having an amino acid sequence registered as accession number O88038 in UniProtKB/Swiss-Prot. It can be a homologue, such as an orthologue thereof.
  • the SapB protein was discovered by Welly et al. (Cell 1991;65(4):641-650) and subsequently shown to contain dehydroalanine and lanthionine linkages (Proc Natl Acad Sci U S A. 2004;101(31):11448-53). This modified peptide can be fully synthesized (J. Org. Chem. 2018; 83(14): 7528-7533).
  • the amino acid sequence registered as accession number O88038 in UniProtKB/Swiss-Prot is MNLFDLQSME TPKEEAMGDV ETGSRASLLLL CGDSSLSITT CN (SEQ ID NO: 1), and the amino acid sequence of the 22nd to 42nd regions is TG SRA SLLC .
  • GD S SL S ITT CN (SEQ ID NO: 2) ⁇ wherein the underlined amino acids have chemical modifications and the S at position 3 and C at position 10 are linked by a lanthionine bond between the ⁇ carbons , S at position 13 and C at position 20 are linked by a lanthionine bond between ⁇ carbons, and S at position 6 and S at position 16 are dehydroalanine ⁇ .
  • SapB can be obtained, for example, by introducing a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 into a microorganism (eg, bacteria) such as actinomycetes and expressing it.
  • the SapB protein may have surfactant activity (Mol. Microbiol. 1998;30(3):595-602).
  • SapB is part of RamS (SapB precursor) as described above and is encoded by the ramS gene.
  • the ramS gene resides within an operon that includes ramC, ramS, ramA, ramB, and ramR.
  • ramC, ramS, ramA, and ramB can be driven by promoter ramCSABp, and ramR can be driven by promoter ramRp. Therefore, the SapB protein may be produced by introducing the operon into a microorganism (eg, bacteria) such as an actinomycete (eg, Streptomyces).
  • a microorganism
  • SapB operon region-integrating cells cells introduced with the SapB operon are referred to as SapB operon region-integrating cells.
  • the SapB operon can be a SapB operon that is foreign or heterologous to the introduced cell.
  • the SapB protein may be produced by introducing only the ramS gene into a microorganism (eg, bacterium). Of course, it can also be obtained by chemical total synthesis.
  • Modified SapB protein with novel modification According to the present disclosure, modifications of the SapB protein are provided. Modified forms of the SapB protein have been isolated. In addition, modified SapB proteins have novel chemical modifications.
  • the modified isolated SapB protein has a modified or protected amino group.
  • the amino group is, in a preferred embodiment, the amino group of the N-terminal amino acid.
  • the amino group in a preferred embodiment, can be the amino group attached to the ⁇ -carbon of the N-terminal amino acid.
  • the SapB protein can be derived from microorganisms.
  • the SapB protein may be derived from Streptomyces.
  • the SapB protein may be derived from an actinomycete of the genus Streptomyces.
  • the SapB protein can be derived from any of the microorganisms (eg, bacteria or actinomycetes) in Table 1 below.
  • the SapB protein is No. 1-100. In one aspect, the SapB protein is no. 1-38 and 41. In one aspect, the SapB protein is no. 1-31. In one embodiment, no. It can be from either 1-20 and 22-25. In one aspect, the SapB protein is no. It can come from anywhere from 1-12 and 20. In these embodiments, the SapB protein can be produced in the corresponding bacteria of the same number.
  • the SapB protein is: coelicolor, S.; griseus, S.; albus, S. scabies, and S. avermitilis from any Streptomyces actinomycete.
  • the SapB protein is any of the amino acid sequences of SEQ ID NOs: 2-9 (especially SEQ ID NOs: 2-7, preferably SEQ ID NOs: 2, 5, 6, 7, 8, and 9) or have a corresponding sequence.
  • having a sequence corresponding to a certain amino acid sequence means that an amino acid sequence having at least 80% or more, 85% or more, 90% or more, or 95% or more sequence identity is aligned with another amino acid sequence. It means the amino acid sequence of the corresponding region.
  • the SapB protein undergoes post-translational modifications after being translated as a protein.
  • Post-translational modifications include: (a) two amino acids of the SapB protein corresponding to Serine 6 and Serine 16 of SEQ ID NO:2 are converted to dehydroalanine; (b) the two amino acids of the SapB protein corresponding to the 3rd serine and the 10th cysteine of SEQ ID NO:2 form a lanthionine bond between their ⁇ -carbons, and (c) the 13th serine of SEQ ID NO:2. and the two amino acids of the SapB protein corresponding to the 20th cysteine form a lanthionine bond between their ⁇ -carbons.
  • the SapB protein may have any one, two, or preferably all of the modifications (a)-(c) above.
  • the SapB protein has two amino acids of the SapB protein corresponding to Serine 6 and Serine 16 of SEQ ID NO: 2 converted to dehydroalanine, and Serine 3 and Cysteine 10 of SEQ ID NO: 2.
  • a lanthionine bond is formed between the ⁇ carbons of the two amino acids of the SapB protein corresponding to , and two amino acids of the SapB protein corresponding to the 13th serine and the 20th cysteine of SEQ ID NO: 2 form a lanthionine bond between the ⁇ carbons. A bond is formed.
  • the aligned SapB protein when aligned with the coelicolor-derived SapB protein has the amino acids of the SapB protein corresponding to the 6th serine and 16th serine of SEQ ID NO: 2 converted to dehydroalanine, and the 3rd of SEQ ID NO: 2
  • a lanthionine bond is formed between the ⁇ -carbons of the serine and the 10th cysteine in SEQ ID NO: 2
  • a lanthionine bond is formed between the ⁇ -carbons of the 13th serine and the 20th cysteine of SEQ ID NO:2.
  • the SapB protein has an amino acid sequence according to any one of SEQ ID NOs: 2-9 (especially SEQ ID NOs: 2-7, preferably SEQ ID NOs: 2, 5, 6, 7, 8 and 9) and may have one, two, or preferably all of the modifications (a)-(c) above.
  • the modified SapB protein has the amino group linked to the ⁇ -carbon of the N-terminal amino acid chemically modified (also simply referred to as "modified") or protected.
  • This modification or protection may, for example, result in resistance to Edman degradation. Edman degradation, for example, can be performed under conditions suitable for amino acid sequencing.
  • Such N-terminal modifications or protections can be beneficial in improving protein stability.
  • the surface-active and emulsifying effects of modifications of the SapB protein can be retained.
  • the N-terminal modification or protection may be modification or protection with an amide group, and in a more preferred embodiment, modification or protection with an NH 2 -C(O)- group.
  • modifications of the SapB protein of the present disclosure have the structure of formula (I): have
  • compositions are provided that include modifications of the SapB protein according to the present disclosure.
  • compositions can include native SapB proteins and modifications of SapB proteins.
  • the composition may have, for example, a surface tension-lowering, emulsifying, demulsifying, penetrating, dispersing, or anti-redeposition effect. Therefore, the composition can be used as a surfactant.
  • the composition can also be used as an emulsifier.
  • the composition may contain an aqueous solvent (eg, water).
  • the composition may contain excipients (eg, bulking agents, thickeners, pH modifiers, fragrances, osmotic pressure modifiers, salts, etc.) in addition to the aqueous solvent (eg, water).
  • the composition contains a native SapB protein and a modified SapB protein, and although not particularly limited, the abundance ratio (molar ratio) can be 99:1 to 1:99.
  • the abundance ratio of modified SapB proteins can be increased by changing one or more conditions selected from the group consisting of extended incubation time, increased heating temperature, and increased urea concentration.
  • a method of preparing a SapB protein comprising: providing a cell that produces a SapB protein; incubating the cells at or above a first predetermined temperature or in the absence of urea, thereby obtaining (or extracting) SapB protein;
  • a method is provided, comprising:
  • a method of preparing a SapB protein comprising: providing a cell that produces a SapB protein; incubating the cells under a temperature condition below a second predetermined temperature or in the presence of urea, thereby obtaining (or extracting) SapB protein;
  • a method is provided, comprising:
  • a method of preparing a SapB protein comprising: providing a cell that produces a SapB protein; incubating the cells under a temperature condition equal to or higher than a third predetermined temperature or in the presence of urea, thereby obtaining (or extracting) the SapB protein;
  • a method is provided, comprising: That is, in one aspect of the present invention, A method of preparing a SapB protein;
  • the conditions for extracting SapB protein from cells are (a) under a temperature condition equal to or higher than a first predetermined temperature and in the absence of urea; (b) under temperature conditions below a second predetermined temperature and in the presence of urea; or (c) under temperature conditions above a third predetermined temperature and in the presence of urea.
  • extraction conditions are (a) native (unmodified) SapB protein can be recovered.
  • native (unmodified) SapB protein can be recovered.
  • the extraction conditions are (b)
  • native (unmodified) SapB protein can be recovered.
  • the extraction conditions are (c)
  • the modified form of SapB protein can be recovered. If the extraction conditions are higher temperature conditions and in the presence of higher concentrations of urea, the amount of modified SapB protein recovered can be increased.
  • the resulting extract may contain a mixture of native SapB protein and modifications of SapB protein.
  • the extraction condition is (a)
  • the yield of native SapB protein can be increased by extending the extraction time (incubation time).
  • the extraction condition is (c)
  • extending the extraction time (incubation time) can increase the yield and recover the modified SapB protein.
  • the methods of preparing SapB proteins of the present disclosure may provide SapB proteins and/or modifications of SapB proteins.
  • the methods of preparing a SapB protein of the present disclosure can result in a mixture of SapB proteins and modifications of SapB proteins.
  • incubation may preferably be carried out in an aqueous solvent (preferably water).
  • the treatment in the absence of urea can be performed, for example, in a first aqueous solution described below, and the treatment in the presence of urea can be performed, for example, in a second aqueous solution described below. can be done inside. If the extraction conditions are below the second predetermined temperature and in the absence of urea, only native SapB protein can be obtained.
  • the first predetermined temperature, the second predetermined temperature, and the third predetermined temperature are each independently 0 to 10°C, 10°C to 20°C, 20°C to 30°C, 30°C to 40°C, 40°C to 50°C, 50°C to 60°C, 60°C to 70°C, 70°C to 80°C, 80°C to 90°C, 90°C
  • the temperature may be within one or more temperature ranges selected from the group consisting of ⁇ 95°C and 95°C to 100°C.
  • the predetermined temperature can be included in one, two, or three temperature ranges selected from the group consisting of, for example, 30°C to 50°C, 40°C to 60°C, and 50°C to 70°C.
  • the first predetermined temperature and the third predetermined temperature are each independently room temperature, 37° C., 40° C., 50° C., or 60° C., or two temperatures thereof (e.g., room temperature and 40° C., etc.).
  • the second predetermined temperature is 0-10°C, 10°C-20°C, 20°C-30°C, 30°C-40°C, 40°C-50°C, 50°C-60°C, or 60°C. It can be ⁇ 70°C.
  • the first predetermined temperature or the third predetermined temperature (more preferably, the first predetermined temperature and the third predetermined temperature) is a temperature equal to or higher than the second predetermined temperature. obtain.
  • the treatment temperatures of (a) and (c) can be at or above the treatment temperature of (b).
  • the incubation time can be 30 minutes or longer, 60 minutes or longer, 120 minutes or longer, 180 minutes or longer, or 240 minutes or longer.
  • the incubation time (and its lower limit and upper limit) can be appropriately determined according to the yield of the SapB protein and its modified form. In one aspect, the incubation time can be from 30 minutes to 240 minutes.
  • the modified SapB protein can also be obtained by incubating the extracted SapB protein under the conditions of (c) above.
  • the obtained SapB protein and modifications thereof can be purified.
  • the resulting composition comprising SapB protein and modifications thereof can be subjected to dialysis.
  • Dialysis allows solvent exchange. For example, when removing urea from a solution, dialysis can be performed using a solution that does not contain urea as the dialysate.
  • a composition comprising the resulting SapB protein and modifications thereof can be an aqueous solution.
  • the resulting aqueous solution may have an alkaline pH.
  • modified SapB proteins according to the present disclosure can be used to prepare compositions, including emulsions, from water-soluble compositions and fat-soluble compositions.
  • the water-soluble composition may comprise an aqueous solvent.
  • An emulsion can be obtained by mixing a water-soluble composition and a fat-soluble composition in the presence of a modified form of the SapB protein according to the present disclosure.
  • Modifications of the SapB protein can be added to either or both compositions during or prior to mixing the water-soluble and fat-soluble compositions in an amount sufficient for emulsion formation.
  • the SapB protein can be used for emulsion formation like other surfactants.
  • a sufficient amount for emulsion formation is, for example, an amount to achieve a concentration equal to or higher than the critical micelle concentration (CMC) (eg, exceeding the CMC, 1.1 times or more the CMC, 1.2 times or more, 1. 3-fold or higher, 1.4-fold or higher, 1.5-fold or higher, 2-fold or higher, 3-fold or higher, 5-fold or higher, 10-fold or higher, 20-fold or higher, 30-fold or higher, 50-fold or higher, or 100-fold or higher concentration ).
  • CMC critical micelle concentration
  • a sufficient amount for forming an emulsion can be appropriately determined by those skilled in the art.
  • the emulsion can be appropriately formed by those skilled in the art.
  • the concentration of SapB does not prevent precipitation of SapB, but is preferably an amount that does not cause precipitation of SapB.
  • the emulsion can be an O/W emulsion. In one preferred aspect, the emulsion can be a W/O emulsion. In one preferred aspect, the fat-soluble composition may contain a fat-soluble solvent.
  • the water-soluble composition and fat-soluble composition can be food compositions.
  • the water-soluble composition may include, for example, water and the fat-soluble composition may include edible oil.
  • the obtained emulsion can also be used by mixing with other food compositions.
  • the water-soluble composition and the fat-soluble composition can be compositions suitable for cosmetic compositions.
  • the resulting emulsion can also be used by mixing with other cosmetic compositions.
  • the resulting modifications of the SapB protein can be used at concentrations and/or amounts that do not exhibit biotoxicity.
  • water-soluble compositions and fat-soluble compositions can be used for surface treatment.
  • the water-soluble composition and fat-soluble composition can make the surface hydrophilic by applying it to the surface of an object or the like. Accordingly, in the present disclosure, water-soluble compositions and fat-soluble compositions can be used to improve the wettability of surfaces or to render surfaces hydrophilic.
  • water-soluble and fat-soluble compositions can be applied to a hydrophilic surface, such as an object, to reduce the wettability of the surface or render the surface hydrophobic.
  • the water-soluble and fat-soluble compositions of the present disclosure are amphiphilic, and when the surface is hydrophilic, the hydrophilic portion of the active ingredient contacts the surface and exposes the hydrophobic portion. It is for the sake of becoming. Accordingly, in the present disclosure, water-soluble compositions and fat-soluble compositions can be used to render surfaces hydrophobic.
  • the water-soluble and fat-soluble compositions are used in household detergents, such as laundry detergents (including clothes detergents), fabric softeners, fabric finishes, fabric softeners, antistatic agents, hand sanitizers.
  • Bath cleaners or toiletry products
  • Bath cleaners such as disinfectants, kitchen cleaners, household cleaners, furniture cleaners, toothpaste, toothpaste, body shampoo, hand soap, facial cleanser, shampoo, dry shampoo, hair care products such as hair rinse
  • Pharmaceuticals such as topical skin preparations containing drugs, quasi-drugs such as medicated cosmetics, skin care cosmetics such as milky lotions, lotions, creams, serums, sunscreens, and daytime moisturizers; foundations, lipsticks, makeup bases, and eye makeup It can be used in various forms of products such as makeup cosmetics such as shadows and mascaras, and hair care cosmetics such as hair treatments.
  • the present disclosure provides any of the above articles of manufacture comprising a SapB protein or a modified version of a SapB protein.
  • the SapB protein or the modified SapB protein may be contained in the form of an emulsion (eg, W/O emulsion and O/W emulsion).
  • the SapB protein or modifications of the SapB protein may be dissolved in solution.
  • the SapB protein or modifications of the SapB protein may be dispersed in a solution.
  • the water-soluble and fat-soluble compositions of the present disclosure are useful for wetting an object, for penetrating an object, for foaming an object, for lubrication. (e.g., for lubrication between solids), for antistatic, for antirust, for bactericidal or antibacterial purposes.
  • the water-soluble composition and fat-soluble composition contain, in addition to the SapB protein or a modified form of the SapB protein, other surfactants (e.g., cationic surfactants, anionic surfactants, nonionic surfactants, surfactants, amphoteric surfactants, etc.) may be further included.
  • surfactants e.g., cationic surfactants, anionic surfactants, nonionic surfactants, surfactants, amphoteric surfactants, etc.
  • a method of preparing a SapB protein according to the disclosure comprises culturing a cell, such as a microorganism (e.g., a bacterium (e.g., actinomycete)), having a gene encoding a RamS or SapB protein capable of expression to produce a SapB protein.
  • a cell e.g., a microorganism (e.g, a bacterium (e.g., an actinomycete)) comprises a gene expression cassette having part or all of the SapB operon region. In this way, cells that produce SapB can be obtained.
  • a method of preparing a SapB protein according to the present disclosure may include harvesting cells (eg, fungi) from the culture medium.
  • the origin of the SapB protein and the SapB protein-producing cell may be the same or different.
  • a method of preparing a SapB protein according to the present disclosure may comprise incubating cells in an aqueous solution (first aqueous solution).
  • the aqueous solution may be water, eg distilled water.
  • Incubation in an aqueous solution of bacterial cells is carried out at, for example, 0°C or higher, room temperature, room temperature or higher, 30°C or higher, 35°C or higher, 40°C or higher, 50°C or higher, 60°C or higher, 70°C or higher, or 80°C or higher.
  • a method of preparing a modified SapB protein according to the present disclosure may comprise incubating cells in an aqueous solution containing urea (second aqueous solution). Incubation in the aqueous solution containing urea can be performed without incubation in the first aqueous solution or preferably after incubation in the first aqueous solution.
  • the urea concentration in the second aqueous solution is, for example, 0.2M or higher, 0.5M or higher, 1M or higher, 2M or higher, 3M or higher, 4M or higher, 5M or higher, 6M or higher, 7M or higher, or 8M or higher. obtain.
  • the urea concentration in the second aqueous solution in some embodiments, can be, for example, 1-8 M, can be 2-7 M, can be 3-6 M, such as 4-5 M, such as about 4 M, or about It can be 5M.
  • Incubation in the second aqueous solution at elevated temperatures for extended periods of time promotes conversion of the SapB protein to modified forms, as described below.
  • the incubation in the second aqueous solution is carried out at a temperature at which urea does not precipitate, for example, 90°C or less, preferably 80°C or less, 70°C or less, 60°C or less, 50°C or less, 40°C. Thereafter, the reaction can be carried out at 35° C. or lower, 30° C. or lower, preferably at room temperature.
  • the incubation time in the second aqueous solution is, for example, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, e.g. can be ⁇ 20 minutes.
  • SapB protein can be obtained by incubating in the second aqueous solution at low temperature for a short period of time, and a mixture of SapB protein and modified SapB protein can be obtained by incubating at high temperature for a long period of time. can.
  • the SapB protein obtained can optionally be purified. Purification includes filtration through filter paper (eg, Advantech No. 2 and No. 5C) and/or filters to remove solids (eg, bacterial debris, etc.). Contaminants can be removed by filtering. Purification also includes dialysis. Dialysis can replace the solvent with the dialysate.
  • methods according to the present disclosure can prepare SapB proteins or modifications of SapB proteins, or mixtures thereof.
  • the first aqueous solution may comprise or consist of ultrapure water (eg, 18.2 M ⁇ cm aqueous solution, eg, Milli-Q water), or distilled water.
  • the first aqueous solution may further contain additives such as salts.
  • the solvent of the second aqueous solution may comprise or consist of ultrapure water (eg, 18.2 M ⁇ cm aqueous solution, eg Milli-Q water), or distilled water.
  • the second aqueous solution may further contain additives such as salts.
  • a method for preparing a modified SapB protein according to the present disclosure comprises culturing a microorganism (e.g., bacteria (e.g., actinomycete)) having a gene encoding RamS or SapB protein to produce SapB protein. can contain.
  • the microorganism e.g., bacterium (eg, actinomycete)
  • a method of preparing a modified SapB protein according to the present disclosure may comprise recovering the cells from the culture medium.
  • the origin of the SapB protein and the SapB protein-producing cell may be the same or different.
  • a method of preparing modifications of the SapB protein according to the present disclosure can comprise extracting SapB from bacteria that produced the SapB protein in the presence of urea.
  • the urea concentration is, for example, 0.2M or higher, 0.5M or higher, preferably 1M or higher, more preferably 2M or higher, still more preferably 3M or higher, particularly preferably 4M or higher, 5M or higher, 6M or higher, 7M or higher, or 8M. or higher concentrations.
  • the urea concentration in some embodiments, can be, for example, 1-8M, 2-7M, 3-6M, 3-7M, 3-8M, such as 4-5M.
  • Extraction can be performed at room temperature or under heating conditions.
  • the heating conditions can be, for example, temperature conditions of 40° C. or higher, 50° C. or higher, 60° C. or higher, 70° C. or higher, 80° C. or higher, or 90° C. or higher. Heating conditions can be, for example, temperature conditions of 70°C to 90°C, for example, temperature conditions of about 80°C.
  • the resulting modified SapB protein can be purified if desired. Purification includes filtration through filter paper (eg, Advantech No. 2 and No. 5C) and/or filters to remove solids (eg, bacterial debris, etc.). Contaminants can be removed by filtering.
  • Purification also includes dialysis. Dialysis can replace the solvent with the dialysate.
  • modified product obtained by the above method may be simply referred to as modified product.
  • mixtures of SapB protein and modifications thereof may be obtained.
  • the expression cassette contains a gene encoding the SapB protein operably linked to regulatory sequences.
  • the gene encoding the SapB protein is rendered expressible in the cell by operably linking it to regulatory sequences.
  • a control sequence is, for example, a promoter. Any promoter can be used as long as it allows expression of SapB in SapB-expressing cells.
  • the SapB operon inherently contains genes encoding SapB proteins operably linked to regulatory sequences.
  • a person skilled in the art can construct a system suitable for expression of the SapB protein by appropriately modifying the expression cassette.
  • urea is less than 1 wt/wt%, less than 0.1 wt/wt%, or less than 0.01 wt/wt%, or less than the limit of detection in compositions comprising SapB protein or modifications thereof
  • concentrations can be used.
  • Urea in the composition can be removed by conventional methods, eg, by dialysis.
  • the concentration of the SapB protein is, for example, 0.001 wt/wt% or more in terms of the weight of the SapB protein portion.
  • the composition containing the SapB protein or the composition containing the modified form of the SapB protein can be an aqueous composition and can contain a pH buffer.
  • An aqueous composition is a composition comprising water. In some embodiments, water is included as a solvent.
  • a composition comprising a SapB protein, or a composition comprising a modified form of a SapB protein has 5 or more, 6 or more, 7 or more, preferably 8 or more, more preferably 9 or more, even more preferably 10 or more pH. Increasing the pH is preferable because it increases the amount of SapB protein or its modified form that dissolves in an aqueous solution.
  • compositions comprising the resulting SapB protein, or a composition comprising the resulting modification of the SapB protein may contain, but may be free or substantially free of trifluoroacetic acid (TFA).
  • compositions comprising SapB protein, or compositions comprising modifications of SapB protein are free or substantially free of trifluoroacetic acid (TFA).
  • the composition comprises a SapB protein and modifications thereof.
  • the composition comprising the SapB protein or the composition comprising the modified form of the SapB protein comprises a pH buffering agent and has a pH of 8 or higher, more preferably 9 or higher, and even more preferably 10 or higher.
  • the composition is free or substantially free of trifluoroacetic acid (TFA).
  • the composition comprises a SapB protein and modifications thereof.
  • the composition comprising the resulting SapB protein, or the resulting modified form of the SapB protein comprises a pH buffer and has a pH of 8 or higher, more preferably 9 or higher, and even more preferably 10 or higher.
  • SapB protein and/or modifications thereof at a concentration of 0.1 wt/wt% or more, preferably 0.5 wt/wt% or more, more preferably 1 wt/wt% or more in terms of the weight of the SapB protein portion
  • the composition is free or substantially free of trifluoroacetic acid (TFA).
  • the composition comprises a SapB protein and modifications thereof.
  • the concentration of the SapB protein and/or modifications thereof is, for example, 0.1 wt/wt% or more, 0.2 wt/wt% or more, 0.3 wt/wt% or more, 0.4 wt/wt% or more, 0.5 wt/wt% or more, 0.6 wt/wt% or more, 0.7 wt/wt% or more, 0.8 wt/wt% or more, 0.9 wt/wt% or more, 1.0 wt/wt/ % by weight or more, 1.1% by weight or more, 1.2% by weight or more, 1.3% by weight or more, 1.4% by weight or more, 1.5% by weight or more, 1 .6 weight/weight % or more, 1.7 weight/weight % or more, 1.8 weight/weight % or more, 1.9 weight/weight % or more, or 2 weight/weight % or more.
  • the concentrations are calculated only for the dissolved SapB protein and its modifications.
  • the pH of the compositions of the present disclosure can be 11 or less, 10 or less, or 9 or less. In some embodiments, the pH of the compositions of the present disclosure can be 8-11. In some embodiments, the pH of the compositions of the present disclosure can be 8-10. In some embodiments, the pH of the compositions of the present disclosure can be 9-11. In some embodiments, the pH of the compositions of the present disclosure can be 9-10. In some aspects, the pH of the compositions of the present disclosure can be from 8.5 to 10.5. In some aspects, the pH of the compositions of the present disclosure can be from 9 to 10.5.
  • the composition of the present disclosure contains native SapB protein and can have a pH of 8 or higher.
  • the composition of the present disclosure comprises the modified SapB protein described above and can have a pH of 6 or higher. Under this pH condition, the native SapB protein and the above modifications of the SapB protein can exhibit good solubility in aqueous solutions.
  • pH buffers selected from the group consisting of glycine-HCl, acetic acid, phosphoric acid, Tris, and glycine-NaOH can be used.
  • pH buffers selected from the group consisting of BES, MOPS, TES, HEPES, DIPSO, TAPSO, Tricine, POPSO, HEPPSO, Bicine, TAPS, HEPPS, CHES, and CAPS can be used. .
  • glycine-HCl buffer can be used
  • pH 3-6 acetate buffer can be used
  • pH 5-8 phosphate buffer can be used
  • pH 7-9 Tris buffer can be used.
  • pH 8-11 a glycine-NaOH buffer can be used.
  • the SapB protein and the modified SapB protein are dissolved in an aqueous solution in the compositions of the present disclosure.
  • the SapB protein and modifications of the SapB protein are completely dissolved in the aqueous solution in the compositions of the present disclosure.
  • the SapB protein and modifications of the SapB protein are suspended in an aqueous solution.
  • the compositions of the present disclosure are free of SapB protein and SapB protein precipitates.
  • compositions of the present disclosure have surfactant activity. Accordingly, the compositions of the present disclosure can be used as surfactants or emulsifiers.
  • Example 1 Preparation of highly expressing SapB actinomycete
  • the SapB operon region (including ramC, ramS, ramA, ramB and ramR) was cloned from Streptomyces coelicolor, and the fragment was introduced into an expression vector.
  • ramS (having the amino acid sequence registered as O88038 in UniProtKB/Swiss-Prot) encodes the precursor of SapB protein, and SapB is produced by excision from the precursor.
  • SapB A gene that corresponds to RamS in various actinomycetes and is presumed to encode SapB was identified (hereinafter simply referred to as "SapB” or “SapB protein"), and each SapB operon region was introduced into an expression vector in the same manner as described above.
  • Streptomyces lividans 1326 was transformed with the above expression vector having the gene encoding SapB to obtain a transformant (SapB operon region-integrated SapB protein high expression strain).
  • RamS used in this example has the amino acid sequence of the SapB precursor of SEQ ID NO: 1, but the natural structure of the SapB protein has the following structure due to post-translational modification (Proc Natl Acad Sci USA .2004;101(31):11448-11453).
  • amino acid sequences of other SapB precursors undergo similar post-translational modifications.
  • amino acid sequence is represented according to the primary sequence encoded by the gene.
  • a post-translational modification causes the OH group of serine (S) to react with the SH group of cysteine (C), resulting in the same structure as above, where the methyl groups of the two alanine side chains are linked by S. becomes.
  • Dha indicates dehydroalanine
  • the 3rd alanine and the 10th alanine form a lanthionine bridge
  • the 13th alanine and the 20th alanine form a lanthionine bridge.
  • the SapB expression strain was cultured in a test tube (TSB medium (Bacto (trademark) Tryptic Soy Broth (Soybean-Casein Digest Medium manufactured by Becton Dickinson, product number: 211825) 5 mL, thiostrepton 50 ⁇ g/mL) at 28°C. After that, 1000 ⁇ L of the seed culture solution was inoculated into a flask (TSB medium, 100 mL) as the main culture, and shake-cultured at 28° C. for 4 days.
  • TBSB medium Bosto (trademark) Tryptic Soy Broth (Soybean-Casein Digest Medium manufactured by Becton Dickinson, product number: 211825) 5 mL, thiostrepton 50 ⁇ g/mL
  • 1000 ⁇ L of the seed culture solution was inoculated into a flask (TSB medium, 100 mL) as the main culture, and shake-cultured at 28° C. for 4
  • Example 2 Extraction and Recovery of SapB After the main culture, the culture medium was recovered, and the cells were recovered by centrifugation. The SapB protein was extracted from the recovered cells. Since SapB has low water solubility, the extraction operation was performed under the following conditions for the purpose of increasing the extraction amount.
  • (Condition 1) Extraction was carried out by treating the cells in a 4M urea solution (distilled water containing 4M urea) under heating conditions of 80°C. The cells are then stirred in the urea solution for 2 hours; (Condition 2) For extraction, the cells are stirred in distilled water at 80°C for 2 hours, then cooled to room temperature, urea is added to a final concentration of 4M, and the mixture is stirred for 10 minutes; or (Condition 3). Extraction is carried out by contacting the cells with distilled water at 80°C and incubating.
  • the liquid phase was recovered from the stirred urea solution.
  • the recovered urea solution contained SapB protein.
  • Filter paper no. 2 ADVANTECH
  • filter paper no. Filtration was performed with 5C (ADVANTECH), and the filtrate was collected. After that, it was concentrated with an evaporator. Distilled water was used as an external liquid, and dialysis was performed. The resulting concentrate was freeze-dried.
  • the yield When extracted with water, the yield was 8 mg / L (culture medium volume), whereas under condition 1, the yield was 174 mg / L (culture medium volume), and the extraction using a urea solution yielded Yield increased significantly. In condition 2, the yield was 109 mg/L (culture volume), and the yield was greatly increased by heating and subsequent urea treatment at room temperature.
  • the emulsification action of SapB obtained in Example 2 was confirmed. Oil and water were mixed 1:2 in the presence of 1% by weight/weight of the SapB protein obtained under conditions 1 or 2 and stirred. After 30 seconds, an emulsifying effect was observed in the presence of the SapB protein obtained under condition 1, as shown in the left panel of FIG. Thus, it was revealed that the SapB thus obtained exhibits a good surfactant action and is useful as an emulsifier.
  • the protein obtained under Condition 2 was also found to have a surfactant action. In general, when the concentration of the surfactant is sufficiently higher than the CMC, it causes emulsification, and when the concentration slightly exceeds the CMC, it exhibits a demulsifying action.
  • CMC critical micelle concentration
  • Example 3 Analysis of Obtained SapB
  • the two SapB proteins obtained in Example 2 were analyzed by HPLC, LC-MS, LC-MS/MS and N-terminal amino acid analysis.
  • HPLC SapB was dissolved in 100 mM Tris-HCl (pH 8.0) to 0.1 mg/mL. HPLC measurement was performed under the following conditions. Column: Cosmosil 5Ph-AR-300 (4.6 mm ID x 150 mm) Eluent: A solution H 2 O (0.1% TFA) B solution acetonitrile: 2-propanol (3:2) (0.1% TFA) Flow rate: 1mL/min Gradient: 20-50% / 1-31min Detection: 220 nm
  • the urea concentration during heating was set to 1 M, 2 M, or 4 M, and the heating time was set to 0 to 240 minutes, and the product after the reaction was verified by HPLC.
  • the results were as shown in Figure 2B.
  • peaks of modifiers were confirmed in samples heated for 60 minutes or longer.
  • 40-60% of the native SapB protein was converted to the modified form.
  • the natural form of SapB can be efficiently converted to the modified form by directly reacting with a urea solution.
  • NH 2 -C(O)-NH 2 -CH(CH(OH)CH 3 )-CO-derived b ion peak (theoretical value: 145.0613). This indicates that the N-terminal threonine amino group was modified with NH 2 —C(O)—.
  • the protein was subjected to degradation. As degradation, degradation of the N-terminal amino acid by Edman degradation was performed. Edman degradation is a technique used in decoding amino acid sequences, and was carried out by a conventional method. In Edman degradation, amino acid sequences are analyzed by reacting phenylisothiocyanate with an amino acid followed by acid treatment to liberate a phenylthiohydantoin derivative containing the first residue and the second amino acid. .
  • the lyophilized product of native SapB protein was similarly suspended or dissolved in various buffers. At pH 7, the solution was cloudy, and at pH 8, the solution was clear, and no precipitate was observed.
  • the SapB protein is poorly water-soluble and was conventionally dissolved in trifluoroacetic acid (TFA). Although TFA is known as an organic acid, our results show that a TFA-free SapB protein solution can be prepared by dissolving the SapB protein in an alkaline buffer.
  • TFA trifluoroacetic acid
  • the centrifugal supernatant of the buffer solutions having various pHs in which the modified SapB protein obtained above was suspended or dissolved was analyzed by HPLC under the same conditions as in Example 3. The results were as shown in FIG. As shown in FIG. 6, the peak indicated by the arrowhead in the figure was strongly detected at pH 7 or higher. This peak corresponds to a modified form of the dissolved SapB protein. The same peak was also weakly detected at pH 6.
  • Example 4 Hydrophilic Treatment of Surface with SapB Protein A polycarbonate plate having a hydrophobic surface was coated with native SapB protein, and the wettability of the treated surface was tested. Specifically, a freeze-dried native SapB protein was dissolved in a pH 10 buffer solution to 0.1% by weight to obtain an aqueous SapB solution. The SapB aqueous solution was applied to a part of the polycarbonate plate and heated at 60° C. for 5 minutes to evaporate the water, thereby forming a SapB applied processed region. Distilled water was dropped on each of the SapB-untreated area and the SapB-coated area of the polycarbonate plate. As a result, as shown in FIG.
  • SEQ ID NO: 1 S.E.
  • An example amino acid sequence of RamS of coelicolor SEQ ID NO: 2 S. coelicolor.
  • An example amino acid sequence of the SapB protein of S. coelicolor SEQ ID NO: 5 S. coelicolor.
  • An example amino acid sequence of the SapB protein of S. scabies SEQ ID NO: 6 S.
  • An example amino acid sequence of the SapB protein of S. avermitilis SEQ ID NO: 8 S. .
  • avermitilis - source 1..24 > mol_type
  • protein > organism synthetic construct residue: TGGGGASTVS LLSCVSAASV LLCL 24

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